Warburg Syndrome

Warburg syndrome is a congenital oculocerebral disorder. It is caused by a genetic defect that simultaneously affects ocular and cerebral embryogenesis. The characteristic ophthalmic findings reflect the cerebral malformation (agyria or lissencephaly). Two cases, siblings, have been described. The characteristic bilateral ocular findings (leukocoria with microphthalmia) have been discussed and contrasted with simulating entities. Since Warburg syndrome is a lethal disorder, it is important to distinguish these affected infants from those with hydrocephalus with a known better prognosis. Lastly, the early recognition of this autosomal recessive disorder should prompt genetic parental counseling.     

The variable number of tandem repeat polymorphism of platelet glycoprotein Ibalpha and risk of coronary heart disease

Glycoprotein (GP) Ib-IX-V complex plays an important role in formation of platelet-fibrin clot at the area of damaged vessel wall. One polymorphism of GP Ibalpha, the main component of GP Ib-IX-V complex, is due to variable numbers of tandem repeats (VNTRs) in the macroglycopeptide region of this molecule. We studied the association between the presence of different VNTR alleles of GP Ibalpha and the frequency of coronary heart disease (CHD) among individuals recruited to a large community-based case-cohort study (Atherosclerosis Risk in Communities [ARIC] study). We found that the distribution of VNTR alleles of GP Ibalpha is different among whites and African Americans. The B allele (with 3 repeats) of GP Ibalpha is relatively more common among African Americans compared with whites. In African Americans, the CC genotype (homozygous with 2 repeats) is associated with a lower risk of CHD events than all other genotypes.     

Bleeding disorder due to platelet prostaglandin H synthase-1 (PGHS-1) deficiency

Defective platelet prostaglandin H synthase (PGHS) activity has been recognized as a cause of bleeding disorders, but the defect has not been characterized. We evaluated three female patients aged 37, 48 and 55 who presented with a mild bleeding disorder due to platelet dysfunction. None of the patients had underlying diseases or reported use of aspirin or other nonsteroidal anti-inflammatory drugs. Coagulation screening tests and platelet count were normal in each patient. Platelet aggregation in response to adenosine diphosphate (ADP), collagen and epinephrine were subnormal, characterized by an abnormal second-wave aggregation and propensity for disaggregation. Arachidonate-induced platelet aggregation was defective, whereas PGH₂-induced aggregation was normal. Platelet thromboxane A₂ (TXA₂) production in response to arachidonic acid was reduced in all three patients, i.e. 11.7, 4.6 and 4.4 ng TXB₂/3 10⁸ plt respectively (normal range was 49–81 ng/3 10 ⁸ plt), whereas they were normal in response to exogenous PGH₂, i.e. 71.4, 56.6 and 48.9 ng/3 10⁸ plts, respectively (normal range 49–85 ng/3 10⁸ plt). These results are consistent with a deficiency of platelet PGHS activity. The level of the constitutive platelet PGHS-1 and TXA₂ synthase (TXAS) proteins were determined on platelet microsomal fractions by Western blot analysis using affinity-purified polyclonal antibodies highly specific for human PGHS-1 and TXAS, respectively. In two patients the 70 kD PGHS-1 protein was undetectable, whereas it was normal in the third patient. The 60 kD TXAS band was normal in all three patients. These findings indicate that human platelet PGHS-1 deficiency is due to two types of enzyme defects: type 1 defect is manifested by an undetectable PGHS-1 protein in platelets whereas the type 2 defect is manifested by a normal quantity of PGHS-1 protein which has an impaired catalytic activity.     

Sterigmatocystin, 5-Methoxysterigmatocistin,and Their Combinations are Cytotoxic and Genotoxic to A549 and HepG2 Cells and Provoke Phosphorylation of Chk2, but not FANCD2 Checkpoint Proteins

Sterigmatocystin (STC) and 5-methoxysterigmatocystin (5-M-STC) are structurally related mycotoxins with cytotoxic and genotoxic properties. In the present study, we hypothesized that DNA damage induced by non-cytotoxic concentrations of single and combined mycotoxins could alter the phosphorylation of the checkpoint proteins Chk2 and FANCD2 (ELISA) in HepG2 and A549 cells. The cytotoxic potential (MTT test) of single and combined STC and 5-M-STC, the nature of their interaction (additivity, antagonism, or synergy) and DNA damage level (alkaline comet assay) in HepG2 and A549 cells were also investigated. All experiments were performed after 24 h of mycotoxin treatment. 5-M-STC was 10-folds more cytotoxic than STC to both HepG2 and A549 cells. Both mycotoxins are genotoxic to HepG2 and A549 cells by inducing both double and single DNA strand breaks that activate Chk2 (especially in HepG2 cells) but not the FANCD2 protein. STC exerted higher genotoxic potential than 5-M-STC in HepG2 and A549 cells when both toxins were applied individually at the same concentration. Dual combinations of non-cytotoxic mycotoxin concentrations showed additive to antagonizing cytotoxic and genotoxic effects. The absence and low activation of checkpoint proteins during prolonged exposure to non-cytotoxic concentrations of STC and 5-M-STC could support cell proliferation and carcinogenesis.     

Irinotecan toxicity to human blood cells in vitro: relationship between various biomarkers

Toxic effects of the antineoplastic drug irinotecan on human blood cells at concentrations of 9.0 microg/ml and 4.6 microg/ml were evaluated in vitro. Using the alkaline and neutral comet assay significantly increased levels of primary DNA damage in lymphocytes were detected. The induction of apoptosis/necrosis, as determined by a fluorescent assay, was also notably increased. Cytogenetic outcomes of the treatment were assessed by the analysis of structural chromosome aberrations and fluorescence in situ hybridization. A significantly higher incidence of chromatid breaks and complex quadriradials was observed. Painted chromosomes 1, 2 and 4 were equally involved in translocations, but only the chromosome 1 was involved in the formation of quadriradials. Sister chromatid exchange analysis was performed in parallel with the analysis of lymphocyte proliferation kinetics. The higher concentration of irinotecan caused almost seven-time increase, while the lower one caused a five-time increase of the basal sister chromatid exchange frequency, accompanied with significant lowering of the lymphocyte proliferation index. Using the cytokinesis-block micronucleus assay, a dose-dependent increase in micronucleus frequency along with the formation of nuclear buds and nucleoplasmic bridges was noticed. Inhibitory effects of irinotecan on enzyme acetylcholinesterase (AChE) were studied in erythrocytes. An IC(50) value of 5.0 x 10(-7) was established. Irinotecan was found to be strong inhibitor of the acetylcholine hydrolysis and to cause a continuous decrease of catalytic activity of AChE. The results obtained on a single donor may contribute to the understanding of irinotecan toxicity, but further in vitro and in vivo studies are essential in order to clarify remaining issues, especially on possible inter-individual variability in genotoxic responses to the drug.     

Abamectin in the aquatic environment

Abamectin, widely used as a veterinary anthelmintic, medicine against a variety of animal parasites and insects, can runoff from the sites of application and becomes an aquatic pollutant. The aim of this study was to identify the toxicity of abamectin on bacteria, algae, daphnids, and fish. An extremely high toxicity of avermectin to the survival and reproduction of Daphnia magna was observed in 21-day exposure tests. Zebrafish and the algae Scenedesmus subspicatus are less sensitive to avermectin. The compound is expected to have adverse effects on the aquatic environment due to its high toxicity, even at very low concentrations, to daphnids and to fish.     

Genotoxicity of tryptophol in a battery of short-term assays on human white blood cells in vitro

The genotoxic effects of tryptophol (indole-3-ethanol), an aromatic alcohol and known secondary metabolite of the opportunistic yeast Candida albicans and other Candida spp., were studied using a battery of short-term assays on human white blood cells in vitro. The concentration range of tryptophol tested was 0.25 mM to 2.00 mM. Lymphocyte viability and induction of apoptosis/necrosis were studied by simultaneous use of a fluorescent assay with ethidium bromide and acridine orange. Levels of primary DNA damage and dynamics of DNA repair were evaluated using the alkaline comet assay while the levels and nature of residual DNA damage were assessed by the analysis of structural chromosome aberrations, the sister chromatid exchange test and the cytokinesis-block micronucleus assay. The results obtained suggest cytotoxic, cytostatic and genotoxic effects of the tryptophol treatment in vitro that were mainly dose-dependent. The type and the extent of DNA lesions detected in tryptophol-treated samples indicate the possibility that observed damage is mediated by highly reactive aldehyde metabolite and/or free radicals produced by treatment. The results show that mortality of lymphocytes in tryptophol-treated samples was primarily caused by apoptosis. The generation of additional DNA strand breaks and cytogenetic consequences (chromosome aberrations, sister chromatid exchanges and micronuclei), as observed in this study, sustain the possibility that tryptophol toxicity is mediated by the formation of DNA cross-links and aldehyde-protein adducts. In conclusion, this preliminary study elucidates only a part of tryptophol toxicity to human cells. Because current evidence is not sufficient to obtain information relevant for human risk assessment, further in vitro and in vivo studies are essential in order to clarify remaining issues, especially to elucidate the exact mechanisms and nature of the damage produced following treatment as well to estimate possible interindividual variability in genotoxic responses to the chemical.     

Genome damage in oropharyngeal cancer patients treated by radiotherapy

Aim

To estimate genome damage in oropharyngeal cancer patients before, during, and after radiotherapy and to measure the persistence of caused genome damage relevant in the evaluation of secondary cancer risk.

Methods

DNA damage was evaluated in peripheral blood lymphocytes of 10 oropharyngeal cancer patients using alkaline comet assay, analysis of structural chromosome aberrations, and micronucleus assay. Blood samples were taken 2 hours before irradiation on day 1 of the first radiotherapy cycle, 2 hours after the application of the first dose, in the middle of the radiotherapy cycle, within 2 hours after the last received radiotherapy dose, and after 6 and 12 months after radiotherapy.

Results

In most participants, the highest level of primary DNA damage was recorded in blood samples collected after the administration of first radiation dose (mean tail length 25.04 ± 6.23 μm). Most patients also had increased frequency of comets with long tail-nucleus (LTN comets) after the administration of the first radiation dose (mean, 10.50 ± 7.71 per 100 comets), which remained increased in the middle of radiotherapy (mean, 18.30 ± 27.62 per 100 comets). Later on, the levels of primary DNA damage as recorded by the comet assay, slightly diminished. The frequency of structural chromosome aberrations in lymphocytes gradually increased during the radiation cycle (26.50 ± 27.72 per 100 metaphases at the end of the therapy), as well as the frequency of micronuclei (mean total number of micronuclei 167.20 ± 35.69; per 1000 binuclear cells).

Conclusion

Oropharyngeal cancer patients had relatively high levels of primary DNA damage in their peripheral blood lymphocytes even before therapy. The frequency of complex structural chromosome aberrations and the frequency of micronuclei increased with the progression of the radiation cycle and the doses delivered. As the frequency of chromosomal aberrations a year after radiotherapy mostly did not return to pre-therapy values, it represents an important risk factor related to the onset of second cancer.Squamous cell carcinoma of the oropharynx is an uncommon disease, the prevalence of which is about 5%-10% of all newly diagnosed cancer cases in Europe and the United States (1). Pharyngeal carcinomas, like other tumors of the head and neck, are sentinel diseases of exposure to environmental factors. The development of oropharyngeal cancer is strongly associated with tobacco use and alcohol consumption, as well as with the exposure to several occupational carcinogens, vitamin deficiencies, and poor oral hygiene (2). Only a fraction of exposed individuals develop cancer in the head and neck region, which suggests that individual sensitivity to mutagens is an important endogenous risk factor that significantly contributes to the development of the disease (3-5). Although radiation is the mainstay of current therapy for oral cancer, the variability in intrinsic radiosensitivity significantly contributes to the outcome of the disease control. In general, >15% of nasopharyngeal cancer patients develop acute or late symptoms of enhanced radiosensitivity (6). It has been well documented that patients with head and neck malignancies are at considerable risk of developing a second primary tumor either within or outside the head and neck area (7).Apart from the beneficial effect of radiotherapy, adverse consequences on normal tissue are almost always present. Following γ-irradiation, different types of lesions can be detected in the nuclear DNA. DNA single- and double-strand breaks and a plethora of modified nucleotides are induced by direct ionization of DNA and by free radical-mediated reactive oxidative species developed by the radiolysis of water (8). Although unrepaired DNA damage is useful in killing cancerous cells, it can be detrimental to normal cells, leading to the onset of secondary cancer (9). Growing evidence suggests that delayed radiation-induced damage, ie, induced genomic instability may also significantly contribute to the onset of secondary neoplasms (10).The most extensively used biomarkers for the assessment of genotoxic and carcinogenic risks in humans involve cytogenetic endpoints such as chromosomal aberrations, sister chromatid exchanges, and micronuclei in mitogen-stimulated peripheral blood lymphocytes. In the last decade, the alkaline comet assay, as a relatively new biomarker, has also gained an increased application in clinical medicine (11,12). The majority of reports were concerned with either biomonitoring of increased levels of basal DNA damage in cancer patients or excess DNA damage caused by treatment with radiation or antineoplastic drugs (13-15).Background genome damage in oropharyngeal cancer before radiotherapy has never been correlated with the rate of caused genome damage during and elimination of genome damage after radiotherapy. As in most cases, heavy drinkers with oropharyngeal cancer represent a unique population with genome burden related to alcohol consumption and possible genome instability related to cancer. Due to significant improvement of therapy efficiency in this type of cancer, the secondary cancer risk follow-up should be included in medical surveillance of these patients.In this study, the levels of primary and residual DNA/chromosome damage in patients with oropharyngeal cancer were studied using the alkaline comet assay, analysis of structural chromosome aberrations, and cytokinesis-block micronucleus (CBMN) assay in peripheral blood lymphocytes. This study also investigated the susceptibility of cancer patients to radiation doses received in the course of radiotherapy, as well as possible inter-individual differences in the persistence of lymphocyte genome damage one year after irradiation.